This research develops novel, general, and convenient methodologies to characterize ionophores and sensor materials relevant to optical, potentiometric and voltammetric transduction schemes. These ionophore- based chemical sensors are vital components in clinical, physiological, and biomedical environments. The use of polymeric membranes containing two competing ionophores to assess the strength of ion-ionophore interactions directly within the sensing film is established. These measurements are accomplished with an optical and potentiometric technique which are rigorously compared to each other. Influences of different types and concentrations of ionophores, reference ionophores, ionic sites, and membrane matrices/plasticizer are studied to give information on complex formation constants, complex stoichiometry, and extent of ion compatible solvents as a described comparative method. Methods are designed to characterize anion responsive ionophores by utilizing and characterizing electrically charged H+-ionophores. The method is applied to a large selection of established and novel ionophores embedded in a variety of matrices, and the results are compared to the analytical characteristics of the respective chemical transduction scheme. The potentiometric behavior of solid contact membrane electrodes is quantitative characterized as a possible alternative approach to two-ionophore systems. Membrane-internal diffusion potential profiles are directly mapped with an array of modified metal electrodes by interposing them within ion-selective electrode membranes, thereby elucidating the source of possible membrane instabilities and potential drifts.